Analysis of endogenous SRSF2 distribution shows that histone acetylation causes splicing factor depletion from nucleoplasm.

<p>(A) Immunostaining for endogenous SRSF2 in HeLa cells untreated or treated with TSA (0.2 ng/µl, 6 hours). DNA was stained with DAPI. Visualization of endogenous SRSF2 distribution suggests depletion from nucleoplasm after TSA treatment. Scale bars, 10 µm. (B) Total levels of endogenous SRSF2 do not change upon TSA treatment. Box plot showing similar distribution of SRSF2/DAPI intensities in individual untreated or TSA-treated HeLa cells (26 cells of each type). The SRSF2/DAPI ratio is NOT significantly different between CONTROL and TSA cells (Mann-Whitney U test: p = 0.23; Kolmogov-Smirnov test: p = 0.501). (C) TSA treatment causes depletion of nucleoplasmic SRSF2 fraction. Nucleoplasmic intensity was calculated in the same cells as (B) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048084#s3" target="_blank">Materials and Methods</a>). Box plot shows nucleoplasmic/total SRSF2 fractions in CONTROL and TSA cells, which further supports the observation of SRSF2 depletion from nucleoplasm after TSA treatment. The nucleoplasmic SRSF2 fraction is significantly reduced in TSA-treated cells compared to control cells (marked by asterisk; Mann-Whitney U test: <i>p</i> = 0.0017; Kolmogov-Smirnov test: <i>p</i> = 0.018). (D) High resolution imaging analysis of SRSF2 distribution and speckle internal structure in untreated and TSA-treated cells. Control and TSA-treated cells were fixed, immunostained for SRSF2 and analyzed by SIM/OMX microscopy. Upper panels: a representative cell in each condition is shown, where depletion of diffuse SRSF2 in nucleoplasm and concentration in speckles is apparent after TSA treatment. Lower panels: enlarged view of an individual speckle (yellow boxes) from cells in each experimental condition. Scale bars, 5 µm. (E) HP1α knockdown has similar effect than TSA treatment on endogenous SRSF2 distribution. HeLa cells were transfected with control siRNAs or siRNAs against HP1α. After two days, the cells were fixed and stained for endogenous SRSF2. Depletion of nucleoplasmic SRSF2 staining and accumulation in speckles is evidenced, similar to what is seen after TSA treatment (A). Scale bars, 5 µm.